Method of preventing NMDA receptor complex-mediated neuronal damage

ABSTRACT

Disclosed is a method for decreasing NMDA receptor-mediated neuronal damage in a mammal by administering to the mammal a nitroso-compound that generates nitric-oxide or related redox species, in a concentration effective to effect neuroprotection. Also disclosed is a method for decreasing NMDA receptor-mediated neuronal damage in a mammal by administering to the mammal a nitroso-compound that generates nitric oxide (or a related redox species such as NO -   or NO +   equivalent), or a physiologically concentration effective to cause such neuroprotection.

This invention was made with government support under Grant No(s) R01Ey05477 by the NIH. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

This application is a continuation of earlier application U.S. Ser. No.08/482,365, filed Jun. 7, 1995 (to issue as U.S. Pat. No. 5,801,203)which is a continuation-in-part of application U.S. Ser. No. 07/949,342,filed Sep. 22, 1992, issued as U.S. Pat. No. 5,234,956, which in turn isa continuation of U.S. Ser. No. 07/688,965, filed Apr. 19, 1991, nowabandoned. See also Lipton, PCT WO91/02180. Each of the aboveapplications is hereby incorporated by reference.

This invention relates to the treatment of nervous system disorders,particularly disorders mediated by the N-methyl-D-aspartate (NMDA)subtype of excitatory amino acid receptor complex.

Glutamate or related congenors have been implicated as a significantfactor in the neurotoxicity associated with hypoxic-ischemicencephalopathy, anoxia, hypoglycemia, seizures, trauma, and severaldegenerative neurological disorders (Hahn et al., Proc. Natl. Acad. Sci.USA 85:6556, 1988; Choi, Neuron 1:623, 1988; Rothman et al., TrendsNeurosci. 10:299, 1987; Meldrum et al., Trends Pharm. Sci. 11:379,1990). In many central neurons the predominant form of thisneurotoxicity appears to be mediated by activation of the NMDA subtypeof glutamate receptor and subsequent influx of excessive Ca²⁺ (Choi,ibid; Weiss et al., Science 247:1474, 1990). Lei et al. Neuron8:1087-1099 (1992) discloses the use of nitroso-compounds (compoundscontaining the NO group) to treat neurological diseases. Stamler et al.Science 258:1898-1902 (1992) by referencing Lei et al. (1992) alsodiscloses the use of nitroso-compounds to treat neurological diseases.

SUMMARY OF THE INVENTION

Certain compounds protect neurons against NMDA receptor-mediatedneuronal damage. Specifically, nitroglycerin, nitroprusside, and theirnitroso-compound derivatives provide such protection. Thus, one aspectof the invention features a method for decreasing NMDA receptorcomplex-mediated neuronal damage in a mammal by administering one of theabove-described compounds to the mammal, in a concentration effective todecrease such damage.

Without limitation to any particular theory or mechanism of action, itappears that oxidation of the thiol group(s) of the NMDA receptor'sredox modulatory site protect against NMDA receptor-mediated neuronaldamage. It is also known that the active species of nitroglycerin andnitroprusside is nitric oxide or related NO redox species. ¹ See, e.g.,Garthwaite et al. (Trends in Neurosciences 14:60, (1991). One possiblemechanism for the protective effect is NO-induced oxidation of the NMDAreceptor-channel complex, probably mediated by a nitrosation reactioninvolving transfer of the NO group to the thiol(s) of the NMDAreceptor's redox modulatory site, resulting in an RS-NO (NO⁺equivalent). Similarly, the redox species nitroxyl anion (NO⁻) can alsoreact with thiol groups. In contrast, under physiological conditions,NO• (nitric oxide) reacts directly with thiol groups poorly, if at all.

A second aspect of the invention features a method for decreasing NMDAreceptor complex-mediated neuronal damage by administering anitroso-compound, in a concentration effective to causeneuroprotection--e.g., a decrease in such damage. Without wishing to bebound to a specific mechanism of action, it appears that NO or a relatedredox species acts on the thiol group(s) of the redox modulatory site ofthe NMDA receptor-channel complex to protect against NMDAreceptor-mediated damage.

In preferred embodiments of both aspects of the invention, the mammal isa human patient infected with a virus affecting the nervoussystem--e.g., measles or human immunodeficiency virus (HIV). Inparticular, the patient being treated may be infected with HIV and maymanifest symptoms of the AIDS related complex or acquiredimmunodeficiency syndrome (for example neurological manifestations ofHIV (see, e.g., U.S. Ser. No. 571,949), such as those that may betreated according to the present invention, including, but not limitedto, AIDS dimentia complex or cognitive-motor-sensory deficits ofincipient or progressing dementia). Other neuro-degenerative states thatcan be treated according to the invention include Alzheimer's disease,Huntington's disease, amyotrophic lateral sclerosis (ALS or motor neurondisease), Parkinson's disease, neurolathyrism, Guam disease, and thoselisted in table 2, below. Alternatively, the patient may have (or belikely to be subject to) an acute disorder such as hypoxia, anoxia,carbon monoxide poisoning, ischemia, CNS trauma, hypoglycemia, seizures,stroke (including stroke associated with ischemia or subarachnoidhemorrhage), domoic acid poisoning, lead poisoning, or other acutedisorders listed on Table 1, below. Where the patient is likely to besubject to one of the above conditions, the patient could be treatedprophylactically according to the invention. Other diseases mediated (atleast in part) by excitatory amino acid toxicity and can be treated byNMDA receptor complex modulation according to the present invention.Such diseases include: 1) ALS (amyotrophic lateral sclerosis or motorneuron disease); 2) painful types of "peripheral neuropathy" which maybe mediated by excessive glutamate (NMDA) receptor stimulation, e.g.,causalgia and other types of neuropathic pain syndromes, includingpainful types of peripheral neuropathy which may (but need notnecessarily) include a central nervous system component.

By "NMDA receptor-mediated neuronal damage" is meant any neuronal injurywhich is associated with stimulation or co-stimulation of the NMDAreceptor-channel complex, a receptor-channel complex which is found on asubset of mammalian neurons and which includes a binding site for amolecule such as glutamate, NMDA, or similar agonists (see below).Activation of this receptor-channel complex by binding agonists inducesneuronal excitation by opening specific ion channels in the membrane.

By a "nitroso-compound" is meant any compound which produces asufficient amount of NO (most probably a related redox species such asan NO⁺ or NO⁻ equivalent) upon administration to a mammal to decreaseneuronal damage or injury. For convenience, the less precise term"NO-generating compound" is used to include compounds that produce theabove described NO⁻ related redox species (e.g., RS-NO, an NO⁺equivalent, or NO⁻) or a physiologically acceptable salt thereof.

Useful compounds of the second aspect of the invention include anynitroso-compound. Verification that a particular compound providesprotective oxidation of the NMDA receptor itself is a step wellunderstood by those skilled in the art (see, e.g., Lipton, PCT WO91/02810).

The two preferred compounds of the first aspect of the invention (i.e.,nitroglycerin and sodium nitroprusside) provide the advantage of aproven record of safe human administration (i.e., for treatment forcardiovascular disorders). Other nitroso-compounds that can be used inthe method of the invention include: isosorbide dinitrate (isordil);S-nitroso captopril (Snocap); Serum albumin coupled to nitric oxide("SA-NO"); Cathepsin coupled to nitric oxide (cathepsin-NO); tissueplasminogen activator coupled to NO (TPA-NO); SIN-1 (or molsidomine)cation-nitrosyl complexes, including Fe²⁺ -nitrosyl complexes;Nicorandil; S-nitrosoglutathione; NO coupled to an adamantinederivative, including memantine (see U.S. patent application Ser. No.07/934,824 hereby incorporated by reference); S-nitrosothiols includingS-nitrosocysteine; quinones, including pyrroloquinoline quinone (PQQ),ester derivatives of PQQ, or ubiquinone; sydnonimins or nonoates havingthe formula ##STR1## where X is any nucleophile including an amine; andagents which generate an oxidizing cascade similar to that generated byNO² such as α-lipoic acid (thioctic acid and its enantiomers);dihydrolipoate; glutathione; ascorbate; and vitamin E.

Any of the above described nitroso-compounds may be combined with otherredox compounds that facilitate production and maintenance of NO. Forexample, direct NO-generators can be combined with pyroloquinolinequinone (PQQ), a known NMDA redox modulator (see U.S. Pat. No.5,091,391), or PQQ's derivative esters, or other quinones such asubiquinone.

Regarding compounds according to the second aspect of the invention, theability of NO to be transported to and cross cell membranes facilitatestherapies according to the invention.

A third aspect of the invention is based on the recognition that theredox species NO• (nitric oxide containing one free electron) leads toneurotoxicity via formation of peroxynitrite (ONOO⁻) (or itsdecomposition products) by reaction with O₂ •⁻ (see FIG. 7, below fordemonstration). Applicant notes that the literature describes theenzyme, NO synthase, which produces nitric oxide in certain cell types;this enzyme and its role in neuronal function is discussed in, e.g.,Garthwaite et al. (Trends in Neurosciences 14:60, 1991), Hope et al.(Proc. Natl. Acad. Sci. USA 88:2811, 1991), and Dawson et al., (Ann.Neurol. 32:297-311, 1992). According to this third aspect of theinvention, nitric oxide synthase is inhibited to effect neuroprotection.This aspect of the invention features administering inhibitors of nitricoxide synthase to decrease the availability of NO and hence decrease theavailability of neurotoxic peroxynitrite (ONOO⁻). This aspect of theinvention may be combined with the first two aspects of the invention orthe nitric oxide syntase inhibitor may be administered independently totreat neurological manifestations of infection with an HIV, to treatneuropathic pain mediated by NMDA teceptor activity, or to treat ALS.

A fourth aspect of the invention features the recognition thatneuroprotective dosages of nitrosocompounds can lower blood pressure asan undesirable side effect in naive patients, but that it is possible tobuild tolerance to this side effect without losing the desiredneuroprotective effect. Accordingly, the fourth aspect featuresadministering a nitroso compound capable of protecting against NMDAreceptor complex-mediated neuronal injury, continuously over an extendedperiod with gradually escalating dosage, beginning at a dosage levelwhich does not substantially reduce the patient's blood pressure, andincreasing to a later dosage level to systemic tolerance of thecompound. The later dosage level is high enough to substantially reducea naive patient's blood pressure, but the continuous administration ofthe compound builds tolerance to the compound's blood-pressure loweringeffect, so that the later dosage level does not in fact substantiallyreduce the patient's blood pressure.

Nitroglycerin is the preferred compound for the fourth aspect of theinvention. It may be administered by transdermal patch as described indetail below (e.g. a patch having a surface area over 50 cm²).Preferably such administration is continous over a period exceeding 24hours.

It is also useful, when acutely administering a nitroso compoundaccording to the first two aspects of the invention, to co-administer ablood-pressure increasing compound such as dopamine.

A fifth aspect of the invention features the administration ofsuperoxide dismutase (SOD), catalase, or both, to limit neurotoxicity bydecreasing the formation of peroxynitrite from the reaction of NO• withsuperoxide anion (O₂ •⁻). The treatment can be adjunctive with the firsttwo aspects of the invention or it can be used independently,particularly to treat neurological manifestations of infection with HIVor of ALS. Polyethylene glycol (PEG) is used to enhance absorption intothe central nervous system (CNS) and efficacy of SOD and/or catalase. AnSOD mimic, the protein-bound polysaccharide of Coriolus versicolor QUEL,termed "PS-K", may also be effective by parenteral or oral routes ofadministration, especially with PEG to enhance CNS absorption, and suchmimics may be substituted for SOD in this aspect of the invention. SeeKariya et al., Mol. Biother. 4:40-46 (1992); and Liu et al., (1989) Am.J. Physiol. 256:589-593.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

DETAILED DESCRIPTION

The drawings are first briefly described.

Drawings

FIGS. 1-3 are graphs showing NMDA-evoked intracellular [Ca²⁺ ] levelsobserved by fura-2 imaging over time as different redox agents includingNTG are administered to cultured neurons. (See Examples 1 and 2.)

FIG. 4 is a graph showing NMDA-evoked currents (including [Ca²⁺ ]) incortical neurons and the effect of different redox agents including NTG.(See Example 3.)

FIGS. 5A-B are graph showing NMDA-evoked intracellular [Ca²⁺ ] inducedin cortical neurons and the effect of different redox agents includingS-nitrosocysteine (SNOC). (See Example 5.)

FIG. 6A-D are bar graphs showing that sodium nitroprusside (SNP) ornitroglycerin (NTG) prevents NMDA-mediated neurotoxicity.

FIGS. 7A and 7B are bar graphs showing superoxide requirement ofneurotoxicity induced by S-nitrosocysteine (SNOC) or peroxynitrite(ONOO⁻).

The present invention is based on the finding that the compoundsnitroprusside and nitroglycerin decrease NMDA receptor complex-mediatedneuronal damage (see below). This neuroprotection may be due tonitrosation or oxidation of the NMDA receptor at the redox modulatorysite, resulting in NO group transfer to the thiol group(s) of the NMDAreceptor's redox modulatory site to form an RS-NO (NO⁺ equivalent). Thischemical reaction leads to a decrease in NMDA receptor-operated channelactivation by excitatory amino acids (such as NMDA or glutamate) and aconcomitant decrease in intracellular calcium influx and amelioration ofneurotoxicity.

An increased level or effect of one or more glutamate-related compoundsis associated with many neurodegenerative disorders (e.g., those listedabove). In addition to glutamate itself, neuronal injury may result fromstimulation of the NMDA receptor-channel complex by other excitatoryamino acids, such as aspartate, quinolinate, homocysteic acid, cysteinesulfonic acid, cysteine, or from stimulation by excitatory peptides,such as N-acetyl aspartyl glutamate.

Nitroglycerin (1,2,3-propanetriol trinitrate or glyceryl trinitrate,abbreviated NTG or GTN), sodium nitroprusside, and NO-generatingderivatives of either one of those compounds are considered to beparticularly useful in the invention.

Compounds of the second aspect of the invention (i.e., nitroso-compoundsor NO-generating compounds and their derivatives) may be tested forefficacy in decreasing neuronal damage using the assays describedbelow--i.e. in assays of NMDA-evoked ionic current (see, e.g., PCT WO91/02810), in assays of NMDA-evoked increases in intracellular Ca²⁺ (seebelow), or in assays of neuronal cell death (see below). An effectivecompound will cause a decrease in ionic current, intracellular Ca²⁺concentration, or in neuronal cell death, respectively. Compounds mostpreferred in the invention are those which effect the greatestprotection of neurons from NMDA receptor complex-mediated injury, e.g.,that injury resulting from stimulation of the NMDA receptor by NMDA(asshown below) or other excitatory amino acids or stimulation byexcitatory peptides, such as N-acetyl aspartyl glutamate.

Assay for Neuronal Cell Function and Death

To test compounds for their ability to prevent neurotoxicity, neuronalcell death may be assayed as follows. Neonatal cortical neurons wereprepared according to the general method of Snodgrass et al. (1980)Brain Res. 190:123-138; and Rosenberg et al (1988) J. Neurosci.8:2887-2899. Cultures are monitored following a brief exposure (5-30minutes) to 30-100 μM NMDA, or to 5 mM DTT (for 5 minutes) followed by30-100 μM NMDA (for 5-30 additional minutes), and overnight incubation(i.e., 16 to 24 hours). Experiments in vivo suggest that a transientchemical reducing state exists in the brain following stroke; theintroduction of the chemical reducing agent DTT may mimic this reducingenvironment, increasing the similarity of the in vitro assay to the invivo situation. The candidate compound is tested by addition (e.g., in aseries of concentrations ranging from 0.1 nM-10 mM) after DTT treatmentbut before NMDA treatment. The candidate compound can be added forminutes, hours, or even days prior to its washout period. Following NMDAexposure, the cultures are incubated an additional 16-24 h at 37° C. inan atmosphere of 5% CO₂ /95% air. Neuronal cultures are scored for cellsurvival after this overnight incubation because NMDA toxicity is oftendelayed by several hours following NMDA exposure. The ability ofcortical neurons to maintain phase-bright appearance and exclude trypanblue is used as an index of survival (Rosenberg et al., Neurosci. Lett.103: 162-168, 1989).

Measurement of Intracellular Ca²⁺

The concentration of intracellular free Ca²⁺ ([Ca²⁺ ]i) is measured inneonatal cortical neurons by digital imaging microscopy with the Ca²⁺sensitive fluorescent dye fura 2, as follows. The same cortical neuronalcultures as described above are used. During Ca²⁺ measurements, unlessotherwise stated the fluid bathing the neurons consists of Hanks'balanced salts: 137.6 mM NaCl, 1 mM NaHCO₃, 0.34 mM Na₂ HPO₄, 0.44 mMKH₂ PO₄, 5.36 mM KCl, 1.25 mM CaCl₂, 0.5 mM MgSO₄, 0.5 mM MgCl₂, 5 mMHepes NaOH, 22.2 mM glucose, and sometimes with phenol red indicator(0.001% v/v); pH 7.2. NMDA (in the absence Mg⁺⁺), glutamate, and othersubstances are usually applied to the neurons by pressure ejection afterdilution in this bath solution. Neuronal [Ca²⁺ ]i is analyzed with fura2-acetoxy-methyl ester (AM) as described [Grynkiewicz, et al., J. Biol.Chem. 260:3440 (1985); Williams et al., Nature 318:558 (1985); Connor etal., J. Neurosci. 7:1384 (1987); Connor et al., Science 240:649 (1988);Cohan et al., J. Neurosci. 7:3588 (1987); Mattson, et al., ibid, 9:3728(1989)]. After adding Eagle's minimum essential medium containing 10 μMfura 2-AM to the neurons, the cultures are incubated at 37° C. in a 5%CO₂ /95% air humidified chamber and then rinsed. The dye is loaded,trapped, and deesterified within 1 hour, as determined by stablefluorescence ratios and the effect of the Ca²⁺ ionophore ionomycin on[Ca²⁺ ]i is measured. During Ca²⁺ imaging, the cells are incubated in asolution of Hepes-buffered saline with Hanks' balanced salts. The [Ca²⁺]i is calculated from ratio images that are obtained by measuring thefluorescence at 500 nm that is excited by 350 and 380 nm light with aDAGE MTI 66 SIT or QUANTEX QX-100 Intensified CCD camera mounted on aZeiss Axiovert 35 microscope. Exposure time for each picture is 500 ms.Analysis is performed with a Quantex (Sunnyvale, Calif.) QX7-210image-processing system. Since cells are exposed to ultraviolet lightonly during data collection (generally less than a total of 20 s percell), bleaching of fura 2 is minimal. Delayed NMDA-receptor mediatedneurotoxicity has been shown to be associated with an early increase inintracellular Ca²⁺ concentration.

Patch-Clamp Recording

Patch-clamp recordings were performed in the whole-cell configurationgenerally using the procedure described by Hamill et al. (1981) asmodified by Lipton and Tauck (1987); and Aizenman et al. (1988).Patch-clamp pipettes typically contained 140 mM KCl (or 120 mM CsCl and20 mM TEA-Cl), 2 mM MgCl₂, 2.25 mM EGTA, 10 mM HEPES-NaOH (pH 7.2).Redox reagents were administered by superfusion (3.5 ml/min), while NMDAand glycine were always coapplied by puffer pipette.

A compound may be tested for utility in the method of the inventionusing any type of neuronal cell from the central nervous system, as longas the cell can be isolated intact by conventional techniques. Althoughcortical neuron cultures are used above, retinal ganglion cell neurons,spinal cord neurons, cerebellar granular neurons, or any neuroncontaining NMDA receptors (e.g., neurons from other regions of thecortex) may also be used. Such neurons may be prenatal or postnatal.

The following examples illustrate compounds useful in the method of theinvention and their efficacy in reducing neuronal damage. These examplesare provided to illustrate the invention and should not be construed aslimiting.

EXAMPLES 1-4 NTG or SNP Inhibits NMDA-Induced Increases in [Ca⁺⁺ ] inRat Cortical Neurons in Culture.

In this experiment, the effect of NTG on intracellular [Ca⁺⁺ ] increasesinduced by NMDA were followed using digital [Ca⁺⁺ ] imaging techniques,based on the dye fura-2.

Specifically, the model for NMDA-mediated neurotoxicity involvesexposure of cultured rat cortical neurons to NMDA in the presence ofglycine (a co-agonist). NMDA (50 μM) induces an increase in [Ca⁺⁺ ]_(i)(intracellular calcium ion concentration) that is further enhanced afterexposure to the strong reducing agent, dithiothreitol (DTT). Chemicalreduction of the NMDA redox modulatory site thiol group(s) with DTTincreases the neurotoxic effect of NMDA.

Antagonism of NMDA receptor-mediated neurotoxicity by NTG isdemonstrated as follows.

Cortical cultures were derived from embryonic (fetal day 15 or 16)Sprague-Dawley rats as described previously (Dichter, 1978; Rosenbergand Aizenman, 1989). Briefly, following dissociation in 0.027% trypsin,cerebral cortical cells were plated at a density of 4.5×10⁵ per 35 mmdish containing poly-L-lysine-coated glass coverslips in Dulbecco'smodified Eagle's medium with Ham's F12 and heat-inactivatediron-supplemented calf serum (Hy-Clone) at a ratio of 8:1:1. After 15days in culture (when the astrocyte layer had become confluent), thecultures were treated with cytosine arabinoside for 72 hr. The culturemedium was replenished 3 times weekly. Cultures were incubated at 36° C.in a 5% CO₂, 95% air humified atmosphere. The cultures were used forexperiments at room temperature (21° C.-24° C.) approximately 1 monthafter plating. Neurons could be reliably identified by morphologicalcriteria under phase-contract optics, as later confirmed by patch-clamprecording.

To permit physiology experiments (Ca²⁺ imaging or patch clamping) undernormal room air conditions, just prior to a recording session theculture medium was exchanged for a solution based on Hanks' balancedsalts (as defined above with 1.25-2.5 mM CaCl₂). To enhancephysiological responses to NMDA, the saline was nominally Mg²⁺ free andcontained 1 μM glycine, a coagonist required for NMDA receptoractivation (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988).Tetrodotoxin (1 μM) was added to block action potentials and ensuingneurotransmitter release from other neurons onto the cell of interest.The presence of intact neurotransmission might have obfuscated theresults if, for example, high K⁺, kainate, or NMDA caused the release ofa substance from one neuron that in turn acted on the cell beingmonitored.

FIG. 1 shows quantifications of [Ca²⁺ ]_(i) from digital representationsof fura-2 images over time for five cortical neurons in the field beingimaged. DTT, NTG, DTNB (5,5-dithio-bis-2-nitrobenzoic acid) were applied(in that order) for approximately 2 minutes and washed out prior to datacollection of NMDA-evoked [Ca²⁺ ]_(i) responses. These data werecollected immediately following exposure to 50 μM NMDA. In FIGS. 1 and2, the NMDA-induced [Ca²⁺ ]_(i) response after DTT pretreatment was setat 100%. Prior to DTT pretreatment, the NMDA-induced [Ca²⁺ ]_(i)response was about 68%. Following DTT, subsequent NTG or SNP treatmentdecreased the NMDA-evoked [Ca²⁺ ]_(i) response to 60% or less. Thestrong oxidizing agent DTNB also decreased the NMDA evoked [Ca²⁺ ]_(i)responses. Subsequent treatment with DTT restored the NMDA-evoked [Ca²⁺]_(i) response to 100%.

EXAMPLE 2

Cortical cultures were prepared as described in Example 1 and werepretreated with DTT, washed, and treated with 100 μM NTG, followed by 50μM NMDA. In FIG. 3, NTG persistently decreased the NMDA-evoked [Ca²⁺]_(i) response, and this decrease persisted through repeatedreadministration of NMDA. Readministration of DTT reversed the effect ofNTG.

EXAMPLE 3

The cortical cultures described above were prepared as described inExamples 1 and 2, and patch clamp recordings were performed instead ofdigital Ca²⁺ imaging. Specifically, NMDA (50 μM) was coapplied withglycine (1 μM) from a pneumatic pipette. Such application resulted in aninward current with the cell voltage clamped at a holding potential of-60 mV. A 2 min incubation in DTT (followed by washout) enhanced theNMDA-evoked current. This current was subsequently attenuated followinga 2 min exposure to NTG (and washout). Reapplication of DTT (followed bywashout) increased the current response to its previous value.

FIG. 4 illustrates sequential compilation of the peak NMDA-evokedcurrents for 18 cells following treatment (and subsequent washout) ofeach redox reagent listed on the abscissa. The temporal order ofaddition of the redox reagents is indicated on the abscissa. Values aremean±SEM, normalized to the 50 μM NMDA response observed after exposureto DTT to permit comparison among several cells. Responses to NMDA thatare statistically smaller than those previously obtained directly afterDTT exposure are marked with an asterisk (P<0.001, ANOVA followed byScheffemultiple comparison of means).

A 2 min exposure to DTT (2 mM, followed by washout) increasedNMDA-activated currents, whereas the addition of NTG (500 μM, followedby washout) inhibited the responses. Subsequent incubation in DTNB (500μM for 2 min, followed by washout) decreased the responses slightly.

EXAMPLE 4

The experiment of Example 3 was repeated using 1-5 mM NEM,N-ethylmalemide, an agent known to alkylate sulfhydryl (thiol) groups ofproteins. Following alkylation, neither NTG nor DTNB significantlyaffected the amplitude of NMDA evoked current, indicating that the redoxmodulatory site of the NMDA receptor, the site reacting with the NOgroup, is comprised of a thiol group(s).

EXAMPLE 5 SNOC Decreases NMDA-Evoked [Ca² ]_(i) Responses

S-nitrosocysteine (SNOC) both liberates NO and participates innitrosation (NO⁺ equivalents reacting with protein thiol groups). FIG.5A is a digital representation of fura-2 calcium images as describedabove, for 10 cortical neurons in a single field. Following eachresponse to NMDA, [Ca²⁺ ]_(i) returned to the baseline value within 1min (data not shown for clarity). The data points are connected bydashed lines merely to emphasize the temporal order of addition. In eachcase, 2 mM DTT was applied for ˜2 min and then washed out prior to theapplication of NMDA and data collection. Values are mean +SEM,normalized to the NMDA (50 μM) response obtained after exposure to DTT(maximum response ˜750 nM [Ca²⁺ ]_(i)).

Following maximal chemical reduction with DTT, the NMDA-evoked responsewas increased, but was subsequently inhibited by a 3 min exposure to 100μM S-nitrosocysteine (SNOC). Following washout of S-nitrosocysteine, theNMDA-elicited response recovered only slightly. A second exposure to DTTfully reversed the inhibitory effect of S-nitrosocysteine. Responses toNMDA that are statistically smaller than those obtained directly afterDTT exposure are marked with an asterisk (P<0.01, ANOVA followed byScheffe multiple comparison of means). FIG. 5B shows that pretreatmentwith NEM blocks the effect of SNOC by alkylating thiol group(s), thuspreventing transnitrosation of the NO group to NMDA receptor thiol(s).

EXAMPLE 6 Nitroglycerin (NTG) or Sodium Nitroprusside (SNP) PreventsNMDA Receptor-Mediated Neurotoxicity

Using the neurotoxicity assay described above, the compounds sodiumnitroprusside and nitroglycerin were tested individually for theirability to increase survival of neonatal cortical neurons. The neuronalcells were incubated for 16-24 hours at 37° C. in a humidifiedatmosphere of 5% CO₂ and 95% air.

As shown in FIGS. 6A-6D, surviving neurons are expressed as thepercentage of viable neurons in the control culture dishes in the sameexperiment. The concentrations of drugs were as follows: APV (2 mM), NTG(100 μM), SNP (400 μM), DTT (0.5-2 mM), and reduced Hb (500 μM). Inexperiments with NTG, cultures were exposed to 30 μM NMDA for 30 min;for the SNP experiments, exposure was to 75 μM NMDA for 5 min. For theexperiments with Hb, NTG or SNP was preincubated with a chemicallyreduced, purified preparation of Hb prior to addition to the cultures.Values are mean±SEM for experiments run in triplicate on siblingcultures on separate days (total of 32 experiments). Astericks indicatesignificant difference compared with the value for DTT-NMDA exposure,P<0.05 (ANOVA followed by Scheffe multiple comparison of means.

We found that either NTG or SNP ameliorated neuronal injury engenderedby the addition of NMDA after exposure to DTT (FIGS. 6A and 6B). Thelatter compound was added to produce chemical reduction of the redoxmodulatory site in order to maximize NMDA-activated current, [Ca²⁺ ]_(i)responses, and neurotoxicity (as reported in the literature). The factthat a supramaximally effective concentration of the NMDAreceptor-specific antagonist APV (2 mM) in combination with NTG failedto prevent neuronal cell death to a greater degree than APV alonesuggests that the lethal effects were mediated via the NMDA receptor(FIG. 6B). In addition, exposure to 500 μM reduced hemoglobin (Hb),which complexes NO, did not affect neuronal survival on its own underthese conditions; however, Hb completely inhibited the effect of 100 μMNTG, signifying that the protective action was mediated by NO (FIG. 6C).Reduced Hb (500 μM) also completely inhibited the protective action of400 μM SNP, again suggesting the involvement of NO (FIG. 6D). It islikely that at least a part of the neuroprotective effect of NO isderived from oxidation of the redox modulatory site, since this actionhas previously been shown to attenuate NMDA receptor-mediatedneurotoxicity.

EXAMPLE 7 Superoxide Dismutase (SOD) Plus Catalase Can PreventNeurotoxicity Mediated by Peroxynitrite (ONOO-) formed from the Reactionof Nitric Oxide (NO•) with Superoxide Anion (O•₂ ⁻)

S-nitrosocysteine was added as a source of NO⁻ to cerebrocorticalcultures (Lei et al., (1992) Neuron 8:1087-1099; Stamler et al., (1992)Science 258:1898-1902). Incubation in this S-nitrosothiol (RS-NO)compound produced dose-dependent killing of cortical neurons. Theneurotoxic effect of maximally-lethal concentrations ofS-nitrosocysteine (200 μM) could be prevented by simultaneous additionof SOD and catalase (50 U/ml each) (FIG. 7A). These findings areconsistent with the very rapid liberation of NO• (t_(1/2)(pH 7.4) ofS-nitrosocysteine ˜ 30 s) for reaction with endogenous O₂ ·⁻ to formperoxynitrite (ONOO⁻) with subsequent neuronal damage.

To test whether it is indeed peroxynitrite (and/or its decompositionproducts) that is neurotoxic, we next showed that purified OONO⁻ killsneurons in a dose-dependent fashion. Peroxynitrite leads to lipidperoxidation and massive oxidation of sulfhydryls (Radi et al., (1991)J. Biol. Chem. 266:4244-4250; Radi et al., (1991) Arch. Biochem.Biophys. 288:481-487). In addition, as predicted if OONO⁻ and/or itsdecomposition products were neurotoxic, reaction with SOD (Ischiropouloset al., (1992) Arch. Biochem. Biophys. 298(2):431-436) and catalase didnot prevent the lethal action of peroxynitrite on neurons (FIG. 7B).These experiments indicate that SOD and catalase might be usefuladjunctive neuroprotective agents that could be administered with thenitroso-compounds, such as NTG and SNP, in order to prevent NMDAreceptor-mediated neurotoxicity, because SOD plus catalase would preventthe formation of neurotoxic ONOO⁻ from any NO• that might be formed bythe addition of the exogenous nitroso-compounds.

The results of the above experiments, as shown in FIG. 7, depict thesuperoxide anion requirement of neurotoxicity induced byS-nitrosocysteine (SNOC) but not peroxynitrite (ONOO⁻). A, B, Superoxidedismutase (SOD, 50 U/ml) plus catalase (cat, 50 U/ml) preventedneurotoxicity induced by SNOC (200 μM, B). Neuronal survival was notsignificantly affected in the presence of SOD/catalase alone compared tothat observed in sibling control cultures. As an additional control, 200μM SNOC that had been incubated at room temperature for several days, inorder to release all NO species, did not result in neurotoxicity (A, farleft columns, labeled "old SNOC"). Values are expressed as mean+s.e.m.(n=9). Statistical comparisons were performed by an analysis of variancefollowed by a Scheffe multiple comparison of means (*, P<0.01 comparedto control; **, P<0.05 compared to control; †, P<0.01 compared tonitroso compound+SOD/catalase).

Specifically, the data in FIG. 7 were generated using mixed neuronal andglial cortical cultures from neonatal rats, as described previously(Lei, et al., (1992) Neuron 8:1087-1099). In at least three separateexperiments, triplicate cultures were incubated overnight in Earle'sbuffered saline solution (EBSS) containing various concentrations ofSNOC, peroxynitrite, SOD, catalase, or glutamate. Similar results wereobtained, however, with 20 min incubations in peroxynitrite, consistentwith the known rapid effects of this potent oxidizing agent. The culturefluid was assayed for lactase dehydrogenase (LDH) as an indicator ofneuronal survival by measuring the absorbance at 450 nm (Koh et al.,(1987) J. Neurosci. Meth. 20:83-90). The cultures were also scored forneuronal viability by cell counting, generally with 0.2% trypan blue,after fixation in 2.5% glutaraldehyde, as described above. Neurons fromcultures grown on 15 mm diameter cover slips were counted in a maskedfashion in ˜30 microscopic fields at 200×. Control cultures scored inthis manner typically contained approximately 1000 viable neurons. Overthe range of values illustrated here, there was a linear relationshipbetween the neuronal cell counts and the LDH viability results, asdetermined by a standard curve based upon experiments with varyingconcentrations of glutamate (0-1 mM). For LDH assays, neuronal survivalwas normalized to that observed in control sibling cultures (100%viability value) and in the presence of 1 mM glutamate (0% viabilityvalue); values slightly below 0% were obtained by extrapolation but werenot significantly different from 0%. Similar experiments on cultures ofpurified astrocytes (lacking neurons) did not produce changes in LDH.

Therapy

To prevent neuronal damage, compounds of the invention may beadministered by any of a number of routes in an amount sufficient toattenuate an NMDA-evoked ionic current or a rise in [Ca²⁺ ]i, orneurotoxicity. The compound may be included in a pharmaceuticalpreparation, using a pharmaceutical carrier (e.g., physiologicalsaline); the exact formulation of the therapeutic mixture depends uponthe route of administration. Preferably, the compound is administeredorally or intravenously, but it may also be administered sublingually,by nasal spray, by transdermal patch, subcutaneously,intraventricularly, intravitreally, or by ointment. The preferredcompounds, nitroglycerin or their derivatives (including all thosepreparations commercially available, e.g., those listed in thePhysician's Desk Reference (1991) under coronary vasodilators or undernitroglycerin or nitroglycerin intravenous and including isosorbidemononitrate, isosorbide dinitrate, nitroglycerin sublingual, Minitran,NT-1, Niotrocor, Nitroderm, Nitrodisc, Nitro-dur, Nitro-Dur II,Nitrofilm, Nitrogard, Nitroglin, Nitropen, Tridil, and6-chloro-2-pyridylmethyl nitrate) are administered at 0.01 mg-60 gm/day,in divided doses. Sodium nitroprusside--Na₂ [Fe(CN)₅ NO]--2H₂ O (fromElkins-Sinn, Inc., Cherry Hill N.J.), Nipride (from Roche, Nutley,N.J.), or other preparations--are administered intravenously at 0.5-10μg/min.

Other nitroso-compounds, determined to be an effective neuroprotectiveagent by the assays described herein, are administered as above, at adosage suitable to reduce neuronal damage, or NMDA evoked ionic currentor increased [Ca²⁺ ]i. Generally, such compounds are administered indosages ranging from 0.01 mg-60 gm/day, more preferably in dosage of0.1-5 mg/day.

Those skilled in the art will understand that there are other factorswhich aid in determining optimum dosage. For example, for NO-conjugateddrugs, the dosage used for the unconjugated drug (e.g. TPA a dosage of0.35-1.08 mg/kg and generally≦0.85 mg/kg) is predictive of usefulNO-conjugate dosage. Dosages may be divided. Treatment may be repeatedas necessary to prevent or alleviate neurological injury. It isdesirable to maintain levels of NO or related redox species in the brainof 1 nM to 500 μM.

For the CNS-protective purposes described herein, nitroso-compounds suchas NTG and SNP can be administered acutely along with pressor agents(e.g., dopamine) to prevent a drop in systemic blood pressure.

With specific reference to nitroglycerin (NTG), patients can be madetolerant to the undesired systemic effects of NTG (e.g. blood pressuredrop, coronary artery dilation or headache), without building toleranceto the desired neuroprotective effect of NTG, e.g., in the brain, spinalchord, and retina. Therefore, it is advantageous to induce NTG toleranceby gradually increasing dosage, thus increasing the neuronal protectiveeffect.

To illustrate the development of systemic tolerance, a human patientcould be made tolerant by intravenous administration of NTG within 18-24hr of continuous infusion (see for example, J. E. Shaffer, B.-A. Han, W.H. Chern, and F. W. Lee, J. Pharmacol. Exper. Therap., 1992;260:286-293;D. C. May, J. J. Popma, Wh. H. Black, S. Scahaffer, H. R. Lee, B. D.Levine, and L. D. Hillis, New Engl. J. Med. 1987;317:805-809; C. M.Newman, J. B. Warren, G. W. Taylor, A. R. Boobis, and D. S. Davis, Br.J. Pharmacol. 1990;99:825-829). Oral, nasal spray, or sublingual NTGcould also be used to induce systemic tolerance.

Another, perhaps easier, method for making humans tolerant within 24hours to the systemic effects of NTG is to administer nitroglycerin asnitropaste or as a transdermal patch for transdermal delivery, forinstance, as follows: 1/2 inch every 4 to 6 hours, while monitoring theblood pressure (to see when hypotension subsides from the appliednitropaste). As tolerated (e.g., in the absence of sudden drops in bloodpressure), the dose of nitropaste is increased to up to 3 inches witheach administration. Under these conditions, tolerance (as evidenced byno effect on systemic blood pressure) will develop in 18-24 hours orless. Nevertheless, the therapeutic effect of NTG on the brain's NMDAreceptors to prevent excitotoxicity should be maintained under theseconditions.

An animal model demonstrating the above-described tolerance follows.

Rat pups were treated with nitroglycerin (NTG) approximately for 36-48hours before performing bilateral carotid ligations. The dosing regimenfor the 3 groups of rat pups used to generate the data was as follows.

Hair was removed from the abdominal region of the rat pup using a razor.One-quarter of a NTG patch (Minitran) was applied to the shaved area. Aquarter patch of this size corresponds to a dose of 0.6 mg/24 hours(whole patch 2.4 mg/24 hr). Between 12 and 16 hours later, anotherquarter-patch was applied to the rat's abdomen, leaving the first on.This procedure was again repeated 12-16 hours later. Each rat pupreceived 3 or 4 quarter patches before surgery, which was performed twodays later and consisted of bilateral carotid ligation followed byhypoxia to induce a stroke. Control animals did not receive NTG patches.

Using this procedure of inducing NTG systemic tolerance, the followingresults were obtained: In the control group, 7/11 (64%) of the animalssuffered large (≧50%) cerebral cortical infarcts. In contrast, in theNTG-treated group, only 2/13 (15%) of the animals had any discerniblecerebral infarct at all (P<0.03 by Fisher's exact test). Thus, NTG wascerebral protective when administered in this fashion.

In more recent trials, the 1/4 patch that is first applied is taken off12-16 hr later, and two new 1/4 patches are applied. 12-16 hr later, thetwo quarter patches are removed, and 3 new (1/4) patches are applied.This may be repeated using 4 new (1/4)=1 whole patch. In this fashion,the dose may be escalated to 2, 3 or more whole patches.

Animals are operated on (to induce a stroke) approximately 48 hr afterthe initial administration of NTG patches.

To convert the dosage given to rat pups in these experiments to a humandose of a NTG patch, the following approximation was used. First, therat weight in grams is converted to surface area in square meters andthe dose of NTG is calculated as dose per square meter. In general, therat pups used here weighed ˜20 grams, equivalent to approximately 5×10⁻³square meters in surface area. Using a standard nomogram, the weight ofa human (e.g., 60 kg) can be converted to square meters (˜2 squaremeters for 60 kg). Thus, there is a scale factor in body area of 1:400when converting rat to human dosage. In addition, the dose for a humanper square meter would be approximately 1/4 the dose per square metergiven to a rat pup. Therefore, the human dose is approximately 100 timesthe rat dose, so that a patch containing 24 mg in the rat experiments isequivalent to a human patch containing about 240 mg. Such a human dosewould have to be more slowly escalated to avoid initial hypotension withthe first few NTG patch applications.

The compounds of the invention can be utilized to protect against anumber of neurotoxic disorders associated with elevated levels oreffects of glutamate or related compounds. Such disorders are mediatedby stimulation of NMDA receptors or by downstream effects of NMDAreceptor overstimulation. These disorders include ischemia, hypoxia,hypoglycemia, trauma to the nervous system, epilepsy, Huntington'sdisease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS),Parkinson's disease, and other neurodegenerative disorders. Inherited oracquired chemical disorders mediated at least in part by NMDA receptoractivation can also be treated. These include hyperammonemia, hepaticencephalopathy, hyperglycinemia, and others. See Tables 1 and 2. Theseinclude hyperammonemia, hepatic encephalopathy, hyperglycinemia andothers (see Tables 1 and 2). Neuropathic pain syndromes such ascausalgia may also be mediated in this manner and can be treated withthe compounds of the invention. The method of the invention isparticularly preferred for the treatment of the AIDS dementia complex(HIV-associated cognitive/motor complex plus incipient forms ofcognitive, motor and sensory deficits not yet fulfilling the stringentcriteria for these complexes) and other neurological manifestations ofthe AIDS virus (HIV-1 or HIV-2). The method may also be used forreduction of neuronal damage resulting from infection with otherviruses, such as measles, which cause damage to the nervous system.Other diseases listed above can be treated.

One aspect of the invention features prolonged administration ofincreasing dosages of NO-generating compounds to establish tolerance ofthe vascular effects (coronary artery dilatation, blood pressure drop,etc.), thereby enabling higher dosages of the compounds forneuroprotection. One particular way to achieve this goal is toadminister the NO-generating compound transdermally, using wellestablished transdermal patch technology. Current transdermalnitroglycerin patches provide about 0.2-0.8 mg/hr. They have a typicalsurface area of 10-30 cm². The standard protocol for the use of suchpatches limits their use to about 12 continuous hours to avoid systemictolerance.

In order to establish systemic tolerance and thereby increase theNO-generating levels available for neuroprotection, a patient is startedon a regime of therapy similar to that currently used for cardiacconditions (e.g., patch loaded with about 2-4 mg nitroglycerin to beused for about 10 hours, or about 0.2-0.4 mg/hr). This regime isfollowed continuously, without any substantial (no more than 4-6 hours)hiatus. Gradually (e.g., after a day), the dosage is increased, andblood pressure is monitored to be sure that systemic tolerance has beenachieved. The rate of increase will depend on the patient, but itgenerally will involve doubling the dosage every 24 hours for a periodof 2-3 days. Typically, levels of about 3-4 mg/hr will be achieved, butdosage could go as high as 2.5 gm/hr. Dosage can be increased byincreasing the loading of existing patches, by increasing the surfacearea of similarly loaded patches, or by increasing the efficiency ofpermeability and the frequency of readministration of the patches. Thoseskilled in the art will appreciate that there are many ways to achievethe goal of systemic tolerance. In one specific method, color codedpatches can be used to reduce the chance for misuse (e.g., red, thenwhite, then blue). The colors would signify the sequence of use of therespective patches. The final patches would be designed to administerlevels of NTG that are higher than any currently available patch canadminister. For example, the final patch may have a size of 50 cm² ormore; or it may be loaded to deliver over 3 mg/hr.

Those skilled in the art will appreciate that tolerance can also beachieved using slow release oral NTG (p.1166 of the 1992 PDR), or usingointments or other transdermal modalities.

Other Embodiments

The method described herein is useful for reducing neuronal injury inany mammal having NMDA receptors. Treatment of neuronal damage in humansis the preferred utility; but the method may also be employedsuccessfully for veterinary purposes. The NO-generating compound may beco-administered with other redox compounds or enzymes to controlsuperoxide (O₂ •⁻)-related damage. Nitric oxide (NO•) is known to reactwith O₂ •⁻ to form peroxynitrite (ONOO⁻) which we have shown is toxic toneurons (see FIG. 7, above). Superoxide dismutase (SOD) plus catalasedecrease the O₂ •⁻ available for this reaction and therefore couldenhance neuroprotection by allowing the NO reaction of nitrosation(transfer of NO⁺ equivalents to thiol groups) to predominate to provideprotection by downregulating the NMDA receptor's redox modulatory site.SOD plus catalase, or similarly acting compounds, can be administeredwith polyethylene glycol to enhance their absorption into the CNS andefficacy (Liu et al.,(1989) Am. J. Physiol. 256:589-593. An SOD mimic,the protein-bound polysaccharide of Coriolus versicolor QUEL, termed"PS-K", may also be effective by parenteral or oral routes ofadministration, especially with PEG to enhance CNS absorption. PQQ(pyrroloquinoline quinone--see U.S. Pat. No. 5,091,391, herebyincorporated by reference or PQQ's derivative esters or other quinonessuch as ubiquinone) could also be useful to accept an electron from NOor from O₂ •⁻ to drive the reaction toward nitrosation with NO+equivalent species and hence toward neuroprotection.

Similarly, other useful agents either by themselves or as adjunctiveagents (to be administered with nitroso-compounds) would limit NOproduction (e.g., nitric oxide synthase (NOS) inhibitors). Suchtreatment would avoid peroxynitrite (ONOO⁻) formation and hence neuronalinjury, e.g., contribution to the AIDS dementia complex and otherneurological manifestations of AIDS. These agents are listed in Table 3(enclosed).

                  TABLE 1                                                         ______________________________________                                        Acute Neurologic Disorders with Neuronal Damage Thought to                    be Mediated at Least in Part by Excitatory Amino Acids*                       ______________________________________                                        i.         domoic acid poisoning from contaminated mussels                    ii.        cerebral ischemia, stroke                                          iii.       hypoxia, anoxia, carbon monoxide poisoning                         iv.        hypoglycemia                                                       v.         prolonged epileptic seizures                                       vi.        mechanical trauma to the nervous system                            vii.       Pb (lead) poisoning                                                ______________________________________                                         *For general reviews, see Choi, Neuron 1:623-34 (1988); and Meldrum and       Garthwaite, Trends Pharmacol. Sci. 11:379-387 (1990).                    

                  TABLE 2                                                         ______________________________________                                        Chronic Neurodegenerative Diseases with Neuronal Damage                       Thought or Proposed to be Mediated at Least in Part by                        Excitatory Amino Acids*                                                       ______________________________________                                        i.      Neurolathyrism-BOAA (β-N-oxa1y1amino-L-                                  alanine) in chick peas                                                ii.     Guam Disease-BMAA (β-N-methyl-amino-L-alanine)                           in flour from cycad seeds                                             iii.    Hungtington's disease                                                 iv.     ALS (amyotrophic lateral sclerosis)                                   v.      Parkinsonism                                                          vi.     Alzheimer's disease                                                   vii.    AIDS dementia complex (HIV-associated                                         cognitive/motor complex)                                              viii.   Olivopontocerebellar atrophy (some recessive                                  forms associated with glutamate dehydrogenase                                 deficiency)                                                           ix.     Hepatic encephalopathy                                                x.      Tourette's syndrome                                                   xi.     Mitochondrial abnormalities and other                                         inherited biochemical disorders                                       a.         MELAS syndrome (mitochondrial myopathy,                                       encephalopathy, lactic acidosis and                                           stroke-like episodes)                                              b.         Rett syndrome                                                      c.         homocysteinuria                                                    d.         hyperprolinemia                                                    e.         hyperglycinemia                                                    f.         hydroxybutyric aminoaciduria                                       g.         sulfite oxidase deficiency                                         ______________________________________                                         *For general reviews, see Choi, Neuron 1:623-34 (1988); and Meldrum and       Garthwaite, Trends Pharmacol. Sci. 11:379-387 (1990).                    

                  TABLE 3                                                         ______________________________________                                        Nitric Oxide Synthase Inhibitors                                              ______________________________________                                        1.       Arginine analogs including N-mono-methyl-L-                                   arginine (NMA)                                                       2.       N-amino-L-arginine (NAA)                                             3.       N-nitro-L-arginine (NNA)                                             4.       N-nitro-L-arginine methyl ester                                      5.       N-iminoethyl-L-ornithine                                             6.       Diphenylene iodonium and analogs                                              See, Steuhr, FASEB J 5:98-103 (1991)                                 7.       Diphenyliodonium, calmodulin inhibitors such                                  as trifluoparizine, calmidazolium chloride                           8.       Phospholipase A.sub.2  inhibitors such as                                     aristolochic acid                                                    9.       Calcineurin inhibitors, e.g., FK-506,                                         cyclosporin A, and analogs including rapamycin                                (inhibit calcineurin and thus nitric oxide                                    synthase by inhibiting its dephosphorylation)                        ______________________________________                                    

What is claimed is:
 1. A method of decreasing NMDA receptor-mediatedneuronal damage in a mammal comprising administering to a patient acomposition comprising a component selected from the group consistingof: superoxide dismuta se (SOD); an SOD mimic; catalase; combinations ofat least two ingredients, said first ingredient being SOD or said mimicand said second ingredient being catalase.
 2. The method of claimwherein said patient is infected with a human immunodeficiency virus(HIV) and said method treats neurological manifestations of HIVinfection.
 3. The method of claim 1 wherein said patient suffers fromamyotrophic lateral sclerosis (ALS) and said method treats neurologicalmanifestations of ALS.
 4. The method of claim 1 wherein said patientsuffers from neuropathic pain mediated by NMDA receptor activity.
 5. Themethod of any one of claims 1-4, wherein said component is formulated inpolyethylene glycol or liposomes to enhance central nervous systemabsorption and efficacy.